CN119506260B

CN119506260B - Tryptophan enzyme mutant and application thereof in indole production

Info

Publication number: CN119506260B
Application number: CN202411728747.9A
Authority: CN
Inventors: 于大禹; 孙磊; 陶福平
Original assignee: Hangzhou Viablife Biotechnology Co ltd
Current assignee: Hangzhou Viablife Biotechnology Co ltd
Priority date: 2024-11-28
Filing date: 2024-11-28
Publication date: 2025-07-25
Anticipated expiration: 2044-11-28
Also published as: CN119506260A

Abstract

The invention discloses a tryptophan enzyme mutant and application thereof in indole production, and relates to the technical field of microbial engineering. The invention carries out amino acid substitution at 389 position of SEQ ID NO. 1 through random mutation and site screening to obtain a new tryptophan enzyme, compared with the existing biosynthesis method, the recombinant bacteria constructed by using the gene segments of the modified tryptophan enzyme mutant can efficiently produce indole, and meanwhile, the indole production method provided by the invention has the advantages of low cost, easily obtained raw materials, greatly reduced production cost, short fermentation period, simple process, high efficiency and NO region limitation. Therefore, the tryptophan mutant and the indole production method provided by the invention have good industrial applicability.

Description

Tryptophan enzyme mutant and application thereof in indole production

Technical Field

The invention relates to the technical field of microbial engineering, in particular to a tryptophan enzyme mutant and application thereof in indole production.

Background

Indigo is an old and classical natural dye derived from indigo plants and has been known for thousands of years. The indigo has excellent color stability and glossiness, is widely applied to the fields of textile dyeing, art drawing, printing and the like, can be used as a medicine raw material, and has biological activities such as antioxidation, antibiosis and the like. The traditional indigo production method mainly comprises two steps, namely, extracting indigo from indigo plants, and preparing the indigo through chemical reaction. The method has high production cost and high environmental load, and the indigo is directly prepared by chemical synthesis. The method has high production efficiency and lower cost, but needs to use chemical reagents harmful to human bodies, and toxic wastewater can be generated in the production process. Compared with the traditional chemical synthesis and plant extraction, the microbial fermentation production of the indigo has the advantages of environmental friendliness, low cost, high product purity and the like. In the future, with further development of technology, microbial fermentation is expected to become a dominant mode of indigo production.

Various microorganisms such as Pseudomonas sp, acinetobacter sp, and Bacillus maxima Bacillus megatreium have been studied for their ability to produce indigo. These microorganisms are capable of catalyzing indole production of indigo by a range of key oxidases, such as naphthalene dioxygenase, styrene dioxygenase, huang Sujia oxidase, and cytochrome P450 monooxygenase, among others. Indole and its homologs and derivatives are widely found in nature, such as auxins, tryptophan, and the like. Indole is mainly used as a raw material of perfume, dye, amino acid and pesticide, but at present, indole is mainly synthesized by one-step synthesis of aniline and ethylene glycol or chemical modes such as separation from coal tar, and at present, high-level fermentation for producing indole is difficult due to cytotoxicity of high-concentration indole, so that a large-scale biosynthesis method is not available.

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims to provide a tryptophan enzyme mutant and application thereof in indole production, and recombinant bacteria constructed by using gene fragments of the tryptophan enzyme mutant can produce indole by taking tryptophan as a substrate, so that the problem that high-concentration indole in the prior art has cytotoxicity and cannot produce indole at a high level is solved.

The invention is realized in the following way:

In a first aspect, the invention provides a tryptophan enzyme TnaA mutant, the amino acid sequence of which is obtained by mutating the amino acid sequence shown in SEQ ID NO.1, wherein the mutation site of the mutant comprises a step of mutating valine 389 into aspartic acid.

In a second aspect, the invention provides a nucleic acid molecule encoding a tryptophan enzyme TnaA mutant as described above.

In a third aspect, the present invention provides a recombinant vector comprising the nucleic acid molecule described above.

In a fourth aspect, the present invention provides a recombinant bacterium comprising the recombinant vector described above.

In a fifth aspect, the invention also provides a construction method of the recombinant bacterium, which comprises the steps of inserting a gene of a tryptophan enzyme TnaA mutant into an expression vector to obtain a recombinant vector, and then introducing the recombinant vector into an original strain to obtain the recombinant bacterium.

In a sixth aspect, the invention also provides application of the tryptophan enzyme TnaA mutant, the nucleic acid molecule, the recombinant vector or the recombinant bacterium in the production of indole and downstream products thereof.

In a seventh aspect, the invention also provides a method for producing indole, which comprises the step of adding the recombinant bacteria into a reaction system containing tryptophan to perform whole-cell catalysis, so that indole can be obtained.

The invention has the following beneficial effects:

the invention is based on tryptophan enzyme shown in SEQ ID NO. 1, amino acid substitution is carried out at 389 th site of SEQ ID NO. 1 through random mutation and site screening, a novel tryptophan enzyme is obtained, compared with the existing biosynthesis method, the novel tryptophan enzyme can efficiently produce indole by utilizing recombinant bacteria constructed by gene fragments of modified tryptophan enzyme mutants, meanwhile, the indole production method provided by the invention has the advantages of low cost, easily obtained raw materials and greatly reduced production cost, and the production method provided by the invention has the advantages of short fermentation period, simple process, high efficiency and NO regional limitation. Therefore, the tryptophan mutant and the indole production method provided by the invention have good industrial applicability.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Tryptophan enzymes (TnaA) can reversibly degrade tryptophan into indole, pyruvic acid and ammonia, and the present invention utilizes this synthetic route to produce indole, but since indole is cytotoxic in high concentrations during microbial fermentation, fermentation of microorganisms is inhibited. In order to overcome the problem, the invention screens tryptophan enzymes from specific sources, mutates the tryptophan enzymes to obtain a tryptophan enzyme mutant, constructs recombinant bacteria from genes of the mutant, and uses tryptophan as a raw material through biocatalysis reaction, so that indole can be efficiently produced, and a new strategy is provided for subsequent production of indigo supply precursor substrates.

In the invention, the tryptophan enzyme TnaA is derived from Providencia rettgeri, the amino acid sequence of the tryptophan enzyme TnaA is shown as SEQ ID NO. 1, and the amino acid sequence of the tryptophan enzyme TnaA mutant obtained based on the tryptophan enzyme TnaA is shown as SEQ ID NO. 3.

The mutant site of the tryptophan enzyme TnaA mutant is 389 th site, and the mutation mode is that valine is mutated into aspartic acid.

Specifically, the preparation method of the tryptophan enzyme TnaA mutant gene comprises the steps of codon optimization of the tryptophan enzyme TnaA gene derived from Providencia rettgeri, PCR amplification of the optimized tryptophan enzyme TnaA gene, dilution of the amplified tryptophan enzyme TnaA gene as a template and error-prone PCR amplification, wherein the amino acid sequence of the optimized tryptophan enzyme TnaA gene is shown as SEQ ID NO. 1, and thus the tryptophan enzyme TnaA mutant gene is obtained.

The recombinant vector provided by the invention is a DNA molecule containing a nucleotide molecule encoding the tryptophan enzyme TnaA mutant or a complementary sequence thereof.

Among them, the vector may be selected from expression vectors conventional in the art, such as pCDFDuet, pET-28a (+), etc.

The recombinant vector can be obtained by adopting the existing preparation method, for example, a gene fragment of a tryptophan enzyme TnaA mutant is obtained by PCR amplification, an expression vector is used as a template, a primer is designed to obtain a linearization vector through inverse PCR amplification, and then the linearization vector is connected with the gene fragment of the mutant through recombinase, so that the recombinant vector inserted into the gene fragment of the tryptophan enzyme TnaA mutant can be obtained.

The recombinant bacterium provided by the invention contains the recombinant vector.

In some embodiments, the host cell of the recombinant bacterium may be a prokaryotic cell or a eukaryotic cell, and the prokaryotic cell may be E.coli, preferably E.coli may be ESCHERICHIA COLIBL (DE 3), ESCHERICHIA COLIDH5 alpha and ESCHERICHIA COLIXL-Blue.

Further, the present invention can provide a whole cell catalyst comprising the recombinant bacterium described above.

The expressed tryptophan enzyme TnaA mutant can efficiently convert tryptophan into indole by using the recombinant strain as a whole-cell catalyst.

The invention also provides a construction method of the recombinant strain, which comprises the steps of inserting the gene of the tryptophan enzyme TnaA mutant into an expression vector to obtain a recombinant vector, and then introducing the recombinant vector into an original strain to obtain the recombinant strain.

Based on the constructed recombinant bacteria, the invention also provides a screening method of the recombinant bacteria, which comprises the steps of selecting the recombinant bacteria into a 96-well plate containing LB culture medium, transferring the cultured bacterial liquid into the 96-well plate containing fermentation culture medium, adding an inducer when the bacterial liquid reaches OD _600nm = 0.6-0.8, diluting the fermentation liquid after fermentation, adding a color reagent, measuring the absorbance of the fermentation liquid, and selecting strains with high absorbance for preservation.

In some embodiments, the recombinant bacteria are cultured in LB medium under the conditions of 37 ℃ and 1000 rpm for 6 h.

In some embodiments, the recombinant bacteria are cultured in a fermentation medium at 30 ℃ under 1000rpm culture 24 h, and the inoculum size of the well plate is 5%.

In some embodiments, the components of the fermentation medium during the screening process include 23.6 g/L yeast powder, 11.8 g/L,K₂HPO₄2.2 g/L,KH₂PO₄9.4 g/L,MgSO₄0.241 g/L,CaCl₂0.011g/L, tryptophan 1 g/L peptone, and glucose 4 g/L.

In some embodiments, the recombinant bacteria are induced to express under the condition that the addition concentration of the inducer IPTG is 0.1 mM and the induction temperature is 30 ℃.

In some embodiments, the dilution ratio of the fermentation broth after the fermentation is finished is 10 times, the adding volume of the color reagent is 10%, and the color reaction time is 10 min.

In some embodiments, the color reagent is prepared by weighing 5g of p-dimethylaminobenzaldehyde in 5mL of ethanol and adding 5mL of concentrated sulfuric acid.

In some embodiments, the absorbance wavelength detected is 568 nm.

The tryptophan enzyme TnaA mutant is obtained through random mutation and screening, and indole and downstream products thereof can be efficiently produced by utilizing the mutant and related biological materials. The present invention can thus also provide a process for the production of indoles based on the mutants described above, which can be applied to different production scenarios, such as shake flask fermentation and fermenter fermentation, depending on the production conditions and production requirements.

Wherein, the shake flask fermentation method comprises the following steps:

(1) Picking recombinant bacteria liquid and streaking to a culture medium flat plate for culture;

(2) Picking the fresh bacterial colony in the step (1) to a shake flask for culturing;

(3) Transferring the bacterial liquid after the culture in the step (2) into a fermentation culture medium for shaking culture;

(4) Adding an inducer at the initial stage of inoculation to perform induction expression;

(5) After fermentation, sampling is carried out to detect the concentration of indole.

In some embodiments, the culture conditions in step (1) are 16 h cultured at 37℃and 220rpm, and the medium used is LB medium.

In some embodiments, the culture conditions in step (2) are culturing at 37℃and 220rpm to logarithmic growth phase, OD _600nm = 5, culturing for 6h, using LB medium, and loading 10%.

In some embodiments, the culture conditions in step (3) are such that when the OD _600nm of the cells pre-cultured in the shake flask reaches about 5, the cells are transferred to a triangular flask containing the fermentation medium (10% of the liquid loading) at 10% of the inoculum size, and fermentation is stopped after 24: 24h under conditions of 30℃and 220 rpm.

Wherein the fermentation medium comprises 23.6 g/L yeast powder, 11.8 g/L,K₂HPO₄2.2 g/L,KH₂PO₄9.4 g/L,MgSO₄0.241 g/L,CaCl₂0.011 g/L, tryptophan 3 g/L peptone, 40 g/L glucose and 20% dibutyl sebacate.

In some embodiments, an inducer is added at the time of culturing to OD _600nm = 0.6-0.8 in step (4), the inducer is 0.1mM IPTG, the induction conditions are 30 ℃, and 3g/L tryptophan is added as a substrate after induction for 2 hours.

The fermentation method of the fermentation tank comprises the following steps:

(2) Picking fresh recombinant bacterial colonies in the step (1) into shake flasks for culture;

(3) Transferring the bacterial liquid after the culture in the step (2) into a fermentation tank for fermentation;

(4) Adding an inducer to perform induction expression;

(5) Tryptophan supplementation as substrate was initiated after induction.

In some embodiments, the culture conditions in step (2) are culture at 37℃and 220 rpm, and the culture medium used is LB medium, and the liquid loading is 10%.

In some embodiments, the culture conditions in step (3) are fermentation temperature 30 ℃, ph=7.5, dissolved oxygen 25% -35%, stirring the associated dissolved oxygen, and stopping fermentation after 48 h.

The specific parameters of the fermentation tank are that ammonia water is used for controlling pH=7.5 in the fermentation process, the fermentation temperature is 30 ℃, DO dissolved oxygen is 25% -35%, feeding is carried out according to the residual glucose, tryptophan is added as a substrate after 4 h is induced, and the tryptophan concentration is controlled to be more than 2 g/L.

The components of the fermentation medium comprise 23.6 g/L of yeast powder, 40 g/L of peptone 11.8 g/L,K₂HPO₄2.2 g/L,KH₂PO₄9.4 g/L,MgSO₄0.241 g/L,CaCl₂0.011 g/L, glucose, 0.002 g/L of pyridoxal phosphate, 20% of dibutyl sebacate and 0.5 mL/L of defoamer, wherein the initial liquid loading amount is 2L.

In some embodiments, the induction conditions in step (4) are 37 ℃ culture to OD _600nm = 20, temperature is adjusted to 30 ℃, and induction is performed by adding 0.2 mM IPTG.

The production method can be used for obtaining indole with higher yield by a microbial fermentation method, has simple process and low production cost, is easy to obtain raw materials, and provides a new idea for biosynthesis of indole.

The present invention will be further described with reference to examples, but it should be understood that the scope of the present invention is not limited by the examples.

Example 1

The construction of the expression strain pCDFDuet-TnaA-BL21 is carried out in the following steps:

S1, codon optimization is carried out on a tryptophan enzyme gene TnaA from Providencia rettgeri, and the sequence of the tryptophan enzyme gene TnaA is shown as SEQ ID NO. 2;

s2, amplifying the optimized TnaA gene by PCR, wherein the nucleotide sequence of the primer is shown as SEQ ID NO. 4 and SEQ ID NO. 5, and the TnaA gene of the target fragment is obtained;

Wherein, the PCR amplification conditions are denatured at 98 ℃, annealed at 55/59/61 ℃, extended at 72 ℃ and cycled 30 times;

S3, carrying out linearization amplification by using a plasmid pCDFDuet-1 as a template through inverse PCR to obtain a linearization expression vector pCDFDuet-1, wherein the required primer nucleotide sequences are shown as SEQ ID NO. 6 and SEQ ID NO. 7;

Wherein, the PCR amplification conditions are denatured at 98 ℃, annealed at 55 ℃, extended at 72 ℃ and cycled 30 times;

s4, connecting a target fragment TnaA with a linearization vector pCDFDuet-1 through a novinay recombinase C112 under the enzyme reaction condition of 37 ℃ and 30min to obtain an expression vector pCDFDuet-TnaA, and transferring the expression vector pCDFDuet-TnaA into competent cells of escherichia coli BL21 through a chemical transformation method.

The primer for PCR amplification according to the present invention has the gene sequences shown in Table 1:

TABLE 1 sequence information

Example 2

The embodiment is constructed by using a random mutation vector pCDF-TnaA, and the specific steps are as follows:

S1, performing error-prone PCR amplification by taking the TnaA gene amplified in the step S2 of the example 1 as a template by diluting 100 times, wherein the nucleotide sequences of the required primers are shown as SEQ ID NO. 4 and SEQ ID NO. 5.

The error-prone PCR system is 100 mu L system, and comprises the following components ：51 μL ddH₂O,10 μL 100 mM Tris-Cl,2.5 μL 2 M KCl,3.5 μL 200 mM MgCl₂,4 μL 25 mM dCTP,4μL 25 mM dTTP,4 μL 5 mM dATP,4 μL 5 mM dGTP,4 μL primer,2 μL 25 mM MnCl₂,1 μL taq DNA polymerase,10 μL template.

PCR amplification conditions were denatured at 98 ℃, annealed at 65-55 ℃, extended at 72 ℃ and cycled 40 times.

S2, after the mutant TnaA gene is obtained, gel recovery and purification are carried out by using a Prmotion biological DNA gel recovery kit.

S3, connecting the target fragment TnaA purified in the step a with the linearization vector pCDFduet-1 obtained in the step C of the example 1 through the novinak recombinase C112, wherein the enzyme reaction condition is 37 ℃ for 30min. The expression vector pCDF-TnaA is obtained and transferred into competent cells of escherichia coli BL21 by a chemical conversion method.

Example 3

The method for rapidly screening the high-yield indole strain by using pCDF-TnaA comprises the following specific steps:

S1, picking fresh pCDFDuet-TnaA and pCDF-TnaA x-BL 21 colonies into 96-well plates containing 400 mu L of LB culture medium in each well, and culturing at 37 ℃ and 1000 rpm for 6h, wherein 2 pCDFDuet-TnaA colonies are picked as a control, and all other 90 wells pick pCDF-TnaA x-BL 21 colonies;

S2, transferring the bacterial liquid after the culture in the step S1 into a 96-well plate containing 760 mu L of fermentation culture medium in each well according to the inoculum size of 5%, and culturing at the culture condition of 30 ℃,1000 rpm and the total fermentation time of 24 h;

s3, adding IPTG with a final concentration of 0.1 mM for induction when culturing to a bacterial body OD _600nm = 0.6-0.8 after inoculation, wherein the induction temperature is 30 ℃;

S4, after fermentation is finished by 24 h, diluting 10 mu L of fermentation liquor sample in 900 mu L of water in each hole, adding 10% of color reagent, placing the mixture at room temperature for reaction 5min, and measuring absorbance at 568 nm wavelength by using an enzyme-labeled instrument after the reaction is finished;

s5, comparing the absorbance values, and preserving the strain with higher absorbance value by using 40% glycerol 1:1.

The formula of the fermentation culture medium adopted is 23.6 g/L of yeast powder, 1 g/L of peptone 11.8 g/L,K₂HPO₄2.2 g/L,KH₂PO₄9.4 g/L,MgSO₄0.241 g/L,CaCl₂0.011 g/L, tryptophan and 4 g/L of glucose.

Example 4

The experimental example is pCDF-TnaA-BL 21 shake flask fermentation verification, and comprises the following specific steps:

1) Picking the pCDF-TnaA x-BL 21 bacterial liquid preserved in the example 3, streaking the bacterial liquid to an LB culture medium plate, and culturing the bacterial liquid at 37 ℃ and 220 rpm to 16 h;

2) Picking the pCDF-TnaA x-BL 21 colony cultured in the step 1) to a 500 mL shake flask with 10% of LB medium liquid loading amount, placing the colony in the shake flask at 37 ℃ and 220 rpm for shake flask culture until OD _600nm is about 5, wherein about 6 hours are needed;

3) Transferring the pCDF-TnaA-BL 21 bacterial liquid after the culture in the step 2) into a 5L shaking flask with the liquid loading amount of a fermentation medium of 10% according to the inoculation amount of 10%, and placing the flask in a shaking flask for culturing at 30 ℃ and 220 rpm until the fermentation is finished, wherein the total fermentation time is 24 hours;

4) Adding IPTG with a final concentration of 0.1 mM when culturing to OD _600nm = 0.6-0.8 after inoculation, and carrying out induced expression at 30 ℃;

5) After induction of 2 h, 3 g/L tryptophan was supplemented as substrate.

The fermentation medium formula is 23.6 g/L yeast powder, 40 g/L peptone 11.8 g/L,K₂HPO₄2.2 g/L,KH₂PO₄9.4 g/L,MgSO₄0.241 g/L,CaCl₂0.011 g/L, glucose and 20% dibutyl sebacate.

The bacterial strain screened in the example 4 is subjected to shake flask fermentation to determine indole yield, plasmids of the bacterial strain with high shake flask fermentation yield are extracted and subjected to sequencing identification, the protein sequence is shown as SEQ ID NO. 3, the mutation site is V389D, and the bacterial strain yield can reach 1.6 g/L at the highest.

Comparative example 1

1) Constructing pCDF-PreTnaA plasmid by taking BL21 as chassis strain and introducing BL21 strain by chemical conversion method, wherein PreTnaA is derived from Providencia rettgeri, and the protein sequence is shown as SEQ ID NO. 1;

2) Shake flask fermentation was performed according to the procedure in example 4;

3) And detecting the indole yield in a liquid phase after fermentation.

Comparative example 2

1) BL21 is taken as a chassis strain, pCDF-PvTnaA plasmid is constructed and introduced into BL21 strain by a chemical conversion method, pvTnaA is derived from protein vulgaris, and the protein sequence is shown as SEQ ID NO. 8;

3) And detecting the indole yield in a liquid phase after fermentation.

Experimental example 1

The fermentation products after the fermentation of example 4 and comparative examples 1 to 2 were subjected to HPLC (high Performance liquid chromatography) to examine the indole yield under the following conditions:

a high performance liquid chromatograph, shimadzu LC-2030Plus;

ShimNex HE C8 chromatographic columns of 4.6X105 mm 3 μm;

mobile phase A is 0.1% acetic acid aqueous solution;

mobile phase B, methanol;

Elution ratio mobile phase a mobile phase b=3:7;

The flow rate is 1 ml/min;

Sample injection amount is 10 mu L;

Column temperature is 35 ℃;

The detection wavelength is 269 nm.

The indole content data for each set are shown in Table 2:

table 2 indole yield data for each group

Example 5

The embodiment is the verification of the fermentation effect of a pCDF-TnaA-BL 21 fermentation tank, and the specific steps are as follows:

Picking single colonies of the high-producing strain pCDF-TnaA x-BL 21 verified in example 4 to 100 mL LB liquid, culturing at 37 ℃ 220 rpm to OD _600nm =2-3 as seed liquid;

Inoculating the seed solution into a 5L fermentation tank containing 2L fermentation medium, culturing at 37 ℃ until OD _600nm = 20, adjusting the temperature to 30 ℃, and adding 0.2mM IPTG for induction;

The components of the fermentation medium are 23.6 g/L of yeast powder, 40 g/L of peptone 11.8 g/L,K₂HPO₄2.2 g/L,KH₂PO₄9.4 g/L,MgSO₄0.241 g/L,CaCl₂0.011 g/L, glucose, 0.002 g/L of pyridoxal phosphate, 20% of dibutyl sebacate and 0.5 mL/L of defoamer;

And (3) parameters of a fermentation tank, namely controlling pH=7.5 by ammonia water in the fermentation process, controlling the fermentation temperature to be 30 ℃, and controlling the DO dissolved oxygen content to be 25% -35%, feeding according to the residual glucose content, adding tryptophan as a substrate after inducing 4 h, and controlling the tryptophan concentration to be more than 2 g/L.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A tryptophan enzyme TnaA mutant is characterized in that the amino acid sequence of the mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO. 1, and the mutation site of the mutant is valine 389 mutated to aspartic acid.

2. A nucleic acid molecule encoding the tryptophan enzyme TnaA mutant of claim 1.

3. A recombinant vector comprising the nucleic acid molecule of claim 2.

4. A recombinant bacterium comprising the recombinant vector according to claim 3.

5. The method for constructing a recombinant bacterium according to claim 4, wherein the recombinant bacterium is obtained by inserting a gene of the tryptophan enzyme TnaA mutant into an expression vector to obtain a recombinant vector and introducing the recombinant vector into a starting strain.

6. The method according to claim 5, wherein the starting strain comprises E.coli.

7. Use of the tryptophan enzyme TnaA mutant of claim 1, the nucleic acid molecule of claim 2, the recombinant vector of claim 3 or the recombinant bacterium of claim 4 in the production of indole.

8. A process for preparing indole, which comprises adding the recombinant bacterium of claim 4 into a tryptophan-containing reaction system for whole cell catalysis.

9. The method according to claim 8, wherein the recombinant bacteria are cultured by streaking the recombinant bacteria on a plate, and then picking up colonies obtained by the culture into shake flasks for expansion culture;

wherein the conditions of the plate culture are that the plate culture is carried out overnight at 37 ℃ and 220 rpm, and the culture medium is LB culture medium;

the conditions of the expansion culture are that the culture is carried out under the conditions of 37 ℃ and 220 rpm, the culture medium is LB culture medium, and the liquid loading amount is 10 percent.

10. The method of claim 9, further comprising fermenting after the expanding culture, wherein the fermenting comprises shake flask fermentation and fermenter fermentation;

inoculating bacterial liquid obtained after amplification culture into a fermentation medium for shake flask culture, and adding an inducer at the initial stage of inoculation for induction expression;

the fermentation in the fermentation tank comprises inoculating the bacterial liquid obtained after the expansion culture into a fermentation medium for fermentation in the fermentation tank, adding an inducer for induction expression, and supplementing tryptophan as a substrate after the induction.

11. The method according to claim 10, wherein the shake flask fermentation is performed under conditions of 30 ℃ and 220 rpm, and the fermentation is stopped after 24: 24 h, and the induced expression is performed under conditions that an inducer is added when the culture is performed until the OD _600nm =0.6-0.8, the inducer is 0.1 mM IPTG, the induction temperature is 30 ℃, and 3: 3 g/L tryptophan is supplemented as a substrate after 2: 2h induction.

12. The method according to claim 10, wherein the components of the fermentation medium in the shake flask fermentation comprise 23.6 g/L yeast powder, 11.8 g/L,K₂HPO₄ 2.2 g/L,KH₂PO₄ 9.4 g/L,MgSO₄ 0.241 g/L,CaCl₂ 0.011 g/L, tryptophan 3 g/L peptone, 40 g/L glucose, 20% dibutyl sebacate.

13. The method according to claim 10, wherein the conditions of the fermentation in the fermenter are that the temperature is 30℃, pH =7.5, the dissolved oxygen amount is 25% -35%, the stirring-related dissolved oxygen is carried out, the fermentation time is 48 h, the conditions of the induction table are that the temperature is 37 ℃ and the culture is carried out until the OD _600nm =20, the temperature is regulated to 30 ℃, 0.2 mM IPTG is added for induction, and 2 g/L or more tryptophan is added as a substrate after 4 hours of induction.

14. The method according to claim 10, wherein the components of the fermentation medium in the fermentation in the fermenter include 23.6 g/L yeast powder, 40 g/L peptone 11.8 g/L,K₂HPO₄ 2.2 g/L,KH₂PO₄ 9.4 g/L,MgSO₄ 0.241 g/L,CaCl₂ 0.011 g/L, glucose, 0.002 g/L pyridoxal phosphate, 20% dibutyl sebacate, and 0.5 mL/L defoamer.